The present invention relates to regulation of gene expression by heparan sulfate or analogs thereof.
Hepadnaviruses, as typified by human hepatitis B virus, the prototype member of this virus family, are small enveloped DNA viruses that produce persistent infections of liver cells and cause acute and chronic hepatitis (Kuroki et al., 1995, J. Biol. Chem. 270:15022-15028). Until recently, the process by which hepatitis B virus particles entered host cells was poorly understood. However, the prior art teaches that human hepatitis B virus binds to cells via a cell receptor protein, i.e., carboxypeptidase D (CPD) which is the human homolog of duck glycoprotein 180 (gp180). Duck gp180, which shares approximately 80% amino acid sequence identity with human carboxypeptidase D, has been demonstrated to serve as the hepatitis B virus receptor which mediates viral entry into cells in a duck hepatocyte infection model (Kuroki et al., 1995, J. Biol. Chem. 270:15022-15028).
Carboxypeptidases perform several important functions in a variety of tissues. These proteins may be broadly divided into two groups based on both function and homology (Skidgel, 1988, Trends Pharmacol. Sci. 9:299-304). One group, which consists of proteins typically 30 to 40 kDa in size, includes the digestive enzymes carboxypeptidase A and carboxypeptidase B. The second group, which includes carboxypeptidase D, is a family of regulatory mammalian basic metallocarboxypeptidases related to ancestral digestive pancreatic carboxypeptidase B. These enzymes, which are typically 50 to 60 kDa in size, specifically cleave C-terminal arginine or lysine residues from peptides and proteins (McGwire et al., 1997, Life Sciences 60:715-724).
The basic metallocarboxypeptidases have a variety of functions such as prohormone processing, activation and inactivation of peptide hormones, regulation of plasminogen binding to cells, and even transcriptional regulation. There are a number of members of the basic metallocarboxypeptidases including the secretory vesicle carboxypeptidase E (CPE, also known as carboxypeptidase H), carboxypeptidase N (CPN), and the extracellular membrane-bound carboxypeptidase M (CPM) (Skidgel, 1988, Trends Pharmacol. Sci. 9:299-304).
Carboxypeptidase D, which was recently isolated and characterized from bovine pituitary membranes, is a 180 kDa membrane-associated protein present in both internal and plasma membranes of the cell, and is believed to play a role in processing of proteins that transit the secretory pathway (Song and Fricker, 1996, J. Biol. Chem. 271:28884-28889). The amino-terminal sequence of bovine membrane CPD is very similar to duck glycoprotein 180 (gp180), a protein identified by its ability to bind hepatitis B virus. More recently, CPD has been identified as a membrane-bound CP present in human skin fibroblasts and in the mouse monocyte macrophage cell line J774A.1 (McGwire et al., 1997, Life Sci. 60:715-724). Prior to these studies, it was understood that the only true membrane-bound CP was CPM, although CPE can associate with membranes through an amphipathic carboxy-terminal sequence.
Mouse and human CPD are similar to bovine pituitary tissue CPD. The gene encoding duck gp180 has also been sequenced (Kuroki et al., 1995, J. Biol. Chem. 270:15022-15028). The deduced amino acid sequence of duck gp180 has three CPE-like domains followed by a predicted transmembrane domain and a short cytoplasmic tail. Glycoprotein 180 has been found to have CP activity and an acidic pH optimum. Based on their similar size, pH optima, and amino-terminal sequence, it appears that bovine CPD is a homolog of duck gp180. Further, comparison of amino acid sequences deduced from full-length cDNA clones demonstrates human CPD is 75% and 90% homologous with duck and rat CPD, respectively.
The distribution of soluble, as well as the membrane-bound form of CPD in rat tissues has been found to be more broad than that of CPE, suggesting that CPD and CPE have functions which are distinct from each other (Song and Fricker, 1996, J. Biol. Chem. 271:2884-2889). Soluble CPD activity has been purified to homogeneity and is characterized as two protein bands of approximately 170 kDa and 135 kDa, which are converted to 155 kDa and 115 kDa by treatment with endoglycosidase F (Song and Fricker, 1996, J. Biol. Chem. 271:2884-2889). The N-terminal amino acid sequences of two soluble forms of CPD were identical to one another and to the predicted N-terminal amino acid sequence of duck gp180. The soluble and membrane bound forms of CPD have similar pH optima, inhibitor specificity, and kinetic parameters for substrate hydrolysis. The highest levels of CPD activity were found in pituitary and adrenal glands, and in brain. Western blot analysis indicated that soluble and membrane bound forms of CPD were present in rat brain, heart, liver, and kidney.
Although the removal of carboxy-terminal basic amino acid residues is required for the activity of many neuropeptides, there may be other receptors such as the insulin receptor which require a similar CP action to produce a functional protein. The possibility that CPD is involved in the processing of growth factors and/or growth factor receptors is supported by studies in Drosophila. More specifically, the Drosophila homolog of CPD, the silver gene, is required for viability, development of cuticular structures, and changes in wing differentiation (Settle et al., 1995, Proc. Natl. Acad. Sci. USA 92:9470-9474). These studies suggest a role for CPD in mammalian cell growth and differentiation.
Although the precise function of CPD and duck gp180 in uninfected cells is not known, duck gp180 shares the greatest homology with carboxypeptidase H which is found on the membranes of secretory granules in many endocrine and neuroendocrine cells and is involved in the post-translational maturation of insulin and enkephalin from their precursor polypeptides (Kuroki et al., supra). Further, carboxypeptidase has other ligands, e.g., CD8.sup.+, besides hepatitis B virus. Indeed, another gp180 has been identified on the surface of human intestinal mucosal cells. Although not extensively studied to date, this human gp180 is reported to bind CD8.sup.+ lymphocytes and may play a role in oral tolerance through an immune suppressor function as suggested by the fact that the level of this gp180 protein is decreased in the mucosal cells of patients with Crohn's disease and with inflammatory bowel disease. Upon treatment with N-glycanase, this gp180 protein, which has not been identified as a CP, migrates at a smaller size on SDS gels than N-glycanase treated CPD. In sum, although carboxypeptidase D, and duck gp180 in avian hepatocytes have been demonstrated to be involved in hepatitis B virus entry into host cells, the normal function of these peptides remains to be elucidated as does the precise mechanism by which these proteins mediate viral entry.
Heparan sulfate proteoglycans have been implicated in virus binding and entry into cells in the case of numerous viruses. See, e.g., Klimstra et al., 1998, J. Virol. 72:7357-7366 (Sindbis virus); Immergluck et al., 1998, J. Gen. Virol. 79:549-559 (herpes simplex virus-1; HSV-1); Trybala et al., 1998, J. Biol. Chem. 273:5047-5052 (pseudorabies virus); Boyle and Compton, 1998, J. Virol. 72:1826-1833 (human cytomegalovirus); Jackson et al., 1996, J. Virol. 70:5282-5287 (foot-and-mouth disease virus); Witrouw and De Clercq, 1997, Gen. Pharmacol. 29:497-511 (human immunodeficiency virus); Chen et al., 1997, Nature Medicine 3:866-871 (dengue virus); Summerford and Samnulski, 1998, J. Virol. 72:1438-1445 (human parvovirus adeno-associated virus type 2); Li et al., 1995, J. Virol. 69:4758-4768 (bovine herpesvirus 1)). In addition, the non-viral human pathogens Chlamydia trachomatis and Neisseria gonorrhoeae, have also been demonstrated to utilize heparan sulfate proteoglycans in their host cell entry mechanism (Herold et al., 1997, Antimicrob. Agents Chemother. 41:2776-2780).
In all of the afore-mentioned studies, heparan sulfate, or its analog heparin, was shown to exhibit anti-viral activity. However, the mechanism of inhibition of virus infection was determined to occur by a physical blocking of the virus interaction with the cell surface, wherein the heparan sulfate either bound to the cellular heparan sulfate proteoglycan (HSP), or bound to the viral proteins capable of interacting with the HSP. Indeed, two heparin-binding proteins, HBNF and MK, were demonstrated to inhibit HSV-1 infection of cells by inhibiting viral adsorption to the cell surface (U.S. Pat. No. 5,461,029). Therefore, the prior art teaches that heparan sulfate and its analogue heparin block virus infection by inhibiting virus adsorption to the host cell surface, which virus adsorption is mediated by virus binding to cell surface heparan sulfate proteoglycans.
In addition to its anti-viral effects, heparin inhibits vascular smooth muscle cell (SMC) proliferation and migration both in vivo and in vitro (Au et al., 1993, Haemostasis 23:177-182). It is not known how heparin produces these effects. However, it appears that heparin's inhibitory action is probably mediated during the G.sub.1 phase of the cell cycle (Au et al., supra, Reilly et al., 1989, J. Biol. Chem. 264:6990-6995). As stated previously herein, the precise mechanism(s) by which heparin inhibits mitogenesis is not known, but heparin has been demonstrated, inter alia, to interfere with various G.sub.1 events, decrease the expression of c-myc and c-fos protooncogenes (Pukac et al., 1990, Cell Regulation 1:435-443) as well as the expression of the Oct-1 gene (Weiser et al., 1997, Mol. Biol. Cell 8:999-1011) and the tissue-type plasminogen activator (t-PA) and collagenase (Au et al., 1993, Haemostasis 23:177-182), inhibit a protein kinase C-dependent pathway for mitogenesis (Castellot et al., 1989, J. Cell Biol. 109:3147-3155), and decrease the number of epidermal growth factor (EGF) receptors on the surface of SMCs (Reilly et al., 1987, J. Cell. Physiol. 131:149-157, Reilly et al., 1988, J. Cell. Physiol. 136:23-32). The prior art does not teach how the number of EGF receptors is decreased, the prior art only discloses that heparin reduces their number by 53-60% without a decrease in the affinity of the receptors for EGF. Further, the prior art discloses that EGF inhibits heparin's antiproliferative effect in SMCs while other mitogens have little or no effect in reducing this effect of heparin (Reilly et al., 1988, J. Cell. Physiol. 136:23-32). In sum, heparin exerts an antiproliferative effect on SMCs although the precise mechanism(s) for this effect remains to be elucidated.
There is an acute and unfilled need for the development of antiviral therapeutics which therapeutics regulate expression of cell surface receptor molecules for the regulation of virus infection of cells. There is also an acute and unmet need for the development of compounds which regulate cell receptor expression per se, as well as an acute need for the identification of compounds which specifically regulate expression of desired target nucleic acids. The present invention satisfies these needs.